1. Field of the Invention
This invention relates to a novel neuropeptide galanin receptor and its nucleic acid sequence.
2. Description of the Related Art
Galanin is a 29 amino acid peptide hormone (30 amino acids in human) which is present in a wide range of central and peripheral tissues. Skofitsch, Peptides 6, 509 (1985); Merchenthaler, Prog. Neurobiol. 40, 711 (1993). Galanin is involved in many diverse physiological functions. Galanin is known to regulate the secretion of both endocrine and exocrine hormones. Galanin inhibits insulin secretion from pancreatic beta cells, and can inhibit pancreatic amylase secretion; in the stomach, galanin inhibits gastrin and somatostatin secretion. Galanin stimulates VIP (vasoactive intestinal protein) release from the hypothalamus, prolactin and growth hormone release from the pituitary; and inhibits the secretion of ACTH in the hypothalamus. Furthermore, the secretion of neurotransmitters can be modulated. For example, galanin can inhibit the release of histamine and norepinephrine in the hypothalamus. Other secondary messenger systems are also regulated: galanin can either stimulate or inhibit intracellular cAMP accumulation; is involved in the closure of N- and L-type voltage-sensitive calcium channels, and in the opening of ATP-sensitive and -insensitive potassium channels; and has been shown to stimulate the release of calcium from intracellular stores. Moreover, galanin is involved in the inhibition of acetylcholine release and the inhibition of muscarinic receptor-mediated phosphoinositide turnover. Bartfai, Crit. Rev. Neurobiol. 7, 229 (1993) Galanin is implicated in the modulation of many cognitive and sensory functions. Galanin has potent antinociceptive effects, and can impair performance in one-trial learning, t-maze, and swim maze learning and memory paradigms. Its inhibition of the anoxic release of glutamate, as well as its inhibitory actions on cholinergic function suggest a role in neuroprotection, and in the development of Alzheimer""s Disease. Crawley, Regulatory Peptides 59, 1 (1995). Galanin is known to induce feeding in rodents and, in contrast with the effects of Neuropeptide Y on feeding, galanin increases preference for fat intake. Akabayashi, Proc. Natl. Acad Sci. USA 91, 10375 (1994). Galanin is also involved in the regulation of gastrointestinal smooth muscle contraction. Because of the important role of galanin in these many physiological processes, there is a strong need to further develop materials and methods for investigating the mechanistic behavior of the receptors and for treating diseased and other abnormal states associated with these physiological processes.
Pharmacological data suggest the existence of several galanin receptor subtypes. Wynick, Proc. Natl. Acad Sci. USA 90, 4231 (1990); Zen-Fa, J. Pharmacol. Exp. Ther. 272, 371 (1995). Galanin receptors are known to be linked to the Gi proteins, and there is some evidence that certain galanin receptor subtypes may be linked to cholera toxin-sensitive Gs proteins. Gillison, Diabetes 43, 24 (1994); Chen, Am. J. Physiol. 266, G113 (1994). One galanin receptor has been cloned and it is a member of the seven transmembrane (7TMD) class of G protein-linked receptors. It has been designated as GalR1. Habert-Ortoli, Proc. Natl. Acad Sci. USA 91, 9780; W095/22608. In addition to this human GalR1 receptor, the GalR1 receptor has been obtained from rat Burgevin, J. Mol. Neurosci. 6, 33 (1995). The in vivo functions mediated through this cloned GalR1 receptor have not yet been elucidated. EP-0711830-A2 disclose a closely-related GalR1 sequence, differing in that Cys15xe2x86x92Trp is varied.
The present invention provides a novel galanin receptor protein, the GalR2 receptor. Also provided are the nucleic acid sequences encoding this novel receptor protein as well as methods for using this protein and its nucleic acid sequence, and methods useful for developing and identifying compounds for the treatment of diseases and disorders in which galanin is implicated. The importance of this discovery is manifested in the effects of galanin, which include antinociceptive activity, smooth muscle contraction, cardiovascular activity, pituitary hormone release, cognition, and increased food intake. Thus, this receptor protein is useful for screening for galanin agonist and antagonist activity for controlling these conditions.
In one aspect of the present invention, we provide isolated nucleic acid sequences for a novel galanin receptor, the GalR2 receptor. In particular, we provide the cDNA sequences encoding the rat receptor. These nucleic acid sequences have a variety of uses. For example, they are useful for making vectors and for transforming cells, both of which are ultimately useful for production of the GalR2 receptor protein. They are also useful as scientific research tools for developing nucleic acid probes for determining receptor expression levels, e.g. to identify diseased or otherwise abnormal states. They are useful for developing analytical tools such as antisense oligonucleotides for selectively inhibiting expression of the receptor gene to determine physiological responses. The present invention can also be used to isolate the homologous nucleic acid sequence of other species, such as human, primate, dog, mouse, etc.
In another aspect of the present invention, we provide a homogeneous composition comprising the receptor GalR2 protein. The protein is useful for screening drugs for agonist and antagonist activity, and, therefore, for screening for drugs useful in regulating physiological responses associated with the GalR2 receptor. Specifically, antagonists to the GalR2 receptor could be used to treat obesity and diabetes by reducing appetite and food consumption, whereas agonists could be used for the treatment of anorexic conditions. Furthermore, drugs could be used to treat Alzheimer""s disease, stroke, neuropathic pain, and/or endocrine disorders. The proteins are also useful for developing antibodies for detection of the protein.
Flowing from the foregoing are a number of other aspects of the invention, including (a) vectors, such as plasmids, comprising the receptor GalR2 nucleic acid sequence that may further comprise additional regulatory elements, e.g, promotors, (b) transformed cells that express the GalR2 receptor, (c) nucleic acid probes, (d) antisense oligonucleotides, (e) agonists, (f) antagonists, and (g) transgenic mammals. Further aspects of the invention comprise methods for making and using the foregoing compounds and compositions.
The invention includes a polynucleotide molecule coding for human GalR2, or a galanin binding fragment thereof A polynucleotide molecule comprising SEQ ID NO:1 or a degenerate variant thereof. A purified and isolated human GalR2 protein. The GalR2 protein comprising SEQ ID NO:2. A purified and isolated human GalR2 protein or fragment thereof having GalR2 binding activity. A polynucleotide molecule coding for a variant of human GalR2 comprising SEQ ID NO:3, or a galanin binding fragment thereof. A polynucleotide molecule comprising SEQ ID NO:3 or a degenerate variant thereof. A purified and isolated variant of human GalR2 protein or fragment thereof having GalR2 binding activity. A purified and isolated variant of human GalR2 protein comprising SEQ ID NO:4.
The foregoing merely summarize certain aspects of the present invention and is not intended, nor should it be construed, to limit the invention in any manner. All patents and other publications recited herein are hereby incorporated by reference in their entirety.